The major histocompatibility complex (MHC) class II locus is strongly associated with the risk of type 1 diabetes, and tissue specificity of the autoimmunity causing diabetes is likely to be determined by the trimolecular complex composed of an MHC molecule, an antigen, and a T cell receptor (TCR). We hypothesize that the interactions between components of this trimolecular complex, all of which are encoded in the germline, determines the nature of T cells involved in the development of type 1 diabetes. Targeting of an insulin B chain 9-23 amino acid peptide (insulin B:9-23) is highly likely to be an essential determinant in the initiation of islet inflammation in the spontaneous diabetes animal model, NOD mouse. We recently discovered that TCRs containing the germline-encoded variable gene (Vgene) sequence called TRAV5D-4 play a critical role to induce anti-islet autoimmunity via the recognition of insulin B:9-23 peptide. Thus, the trimolecular complex consisting of the insulin B:9-23 peptide and TRAV5D-4 TCR alpha chain plays a key role in developing anti-islet autoimmunity; however, how T cells expressing TRAV5D-4 TCRs contribute to the initiation and development of the disease remains to be elucidated. Given evidence that the DQ8 diabetes- susceptible HLA class II molecule is an ortholog of NOD I-Ag7 presenting the insulin B:9-23 peptide and that T cells expressing TRAV13-1 (human ortholog of TRAV5D-4) TCR alpha chains dominantly exist in the pancreas of a type 1 diabetes patient having DQ8, the ultimate goal of this proposal is to verify a proof of concept that insulin targetig by a specific germline-encoded TCR Vgene motif plays a critical role in the development of islet autoimmunity. In this proposal, we aim to determine the molecular mechanism of how TCRs, in particular those containing TRAV5D-4, target the critical peptide, insulin B:9-23, to initiate isle inflammation using the NOD mouse model (Aim 1), and to determine whether T cells expressing TRAV5D-4 are essential for diabetes development in NOD mice (Aim 2). If the development of anti-islet autoimmunity is completely suppressed in the absence of TRAV5D-4, targeting only T cells expressing such essential TRAV genes will enable us to develop a robust immunotherapy with the minimum of side effects. Finally, we will pursue the hypothesis that there is a conceptually similar interaction in the human trimolecular complex consisted of the insulin B:9-23 peptide and TRAV13-1 TCR Vgene motif underlying susceptibility to human type 1 diabetes (Aim 3). Findings from this proposal will provide a deeper understanding of the principles underlying the initiation of islet autoimmunity via the interaction within the insulin trimolecular complex, which will ultimately to be applied to design antigen-based immunodiagnostic and immunotherapeutic clinical studies for type 1diabetes in humans.